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Propositions 1. The presence of proteolytic microorganisms in dairy concentrate production lines contributes to the diversity and spore load of specific thermophiles in end products. (this thesis) 2. The pattern of gene expression relating to the biofilm development is dependent on the circumstance where biofilms are formed but is also largely dissimilar for different biofilm- forming organisms. (this thesis) 3. On the one hand, ‘omics’ data yield novel insights into the cellular inner workings of organisms; on the other hand, the abundance of data also presents many hurdles. 4. The difficulty of translating biological insights to practical use is often underestimated by scientists as well as the general public. 5. There is a need to educate the general public in food safety and nutrition related science. 6. Ideally a PhD study is a personal development trajectory, a process for generating scientific understanding, and a process that delivers practical insights. 7. Success is an accumulation of small steps taken and it is crucial to go in the right direction and not to give up. 8. Acceptance for the ownership of the problem is the first step towards solving a problem. Propositions belonging to the thesis, entitled Thermophilic sporeformers from dairy processing environments Yu Zhao Wageningen, 1 July 2020 Thermophilic sporeformers from dairy processing environments Yu Zhao Thesis committee Promotor Prof. Dr M.H. Zwietering Professor of Food Microbiology Wageningen University & Research Other members Prof. Dr M.A.J.S. van Boekel, Wageningen University & Research Prof. Dr S. Brul, University of Amsterdam Dr M.H.J. Wells-Bennik, NIZO food research, Ede Dr M. van der Voort, Wageningen University & Research This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences) Thermophilic sporeformers from dairy processing environments Yu Zhao Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus, Prof. Dr A. P. J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Wednesday 1 July 2020 at 4 p.m. in the Aula. Yu Zhao Thermophilic sporeformers from dairy processing environments, 160 pages. PhD thesis, Wageningen University, Wageningen, the Netherlands (2020) With references, with summary in English ISBN 978-94-6395-345-0 DOI https://doi.org/10.18174/517519 Table of contents Chapter 1 General introduction 7 Chapter 2 Abiotic and microbiotic factors controlling biofilm 21 formation of thermophilic spore formers Chapter 3 Growth of dairy isolates of Geobacillus 53 thermoglucosidans in skim milk depends on lactose degradation products supplied by Anoxybacillus flavithermus as secondary species Chapter 4 Biofilm dynamics of Geobacillus thermoglucosidans, a 69 dairy processing isolate Chapter 5 Genomic comparison of dairy and non-dairy associated 95 thermophilic sporeformers Chapter 6 General discussion 137 Summary 151 Acknowledgements 153 List of publications 155 Curriculum vitae 157 VLAG graduate school activities 159 Chapter 1 General introduction General introduction 1.1 Dairy powder production Dairy products are an important part of human diet. They contain many nutrients, including high-quality protein, and essential nutrients like minerals (e.g. calcium) and vitamins (e.g. vitamin D), which are considered necessary for homeostasis and therefore good health. Storage of raw milk is challenging since it is prone to microbial spoilage because of its neutral pH, high water content, and high nutrient content. In order to extend its shelf-life, different preservation techniques are applied (Law and Mabbitt, 1983). Powderization, for example, is a widely used method for the preservation of various dairy products, such as whole milk, non-fat milk, and dry buttermilk. Compared to butter, cheese or fluid cow milk products, milk powder consumption is, however, smaller. It is an important diet option for many people, especially for people in the countries where cooling facilities are not widely available. For example, China was the largest whole dry milk consumption area in the world in 2018, and the consumption of whole milk powder takes up 14% of total cow milk consumption (USDA/FAS, 2018). In a nutshell, powderization is about transforming liquid milk into dry powder, which requires removal of (almost) all the water. Two main water removal processes used in milk powder industry are vacuum evaporation and spray drying, which can be further supplemented by other fluid removing technologies such as membrane processes or fluid bed drying (Pisecky, 2012). As an example of a dry powder process, the dairy powder production process in a New Zealand whole milk powder factory, as Scott et al. (2007) described, started with the raw milk’s separation, pasteurization, and standardization. There the raw milk treatment runs were 6-8 hours in length. Raw milk was first preheated using a plate heat exchanger (PHE), and then separated into skim milk and cream. The skim milk and cream were then pasteurized separately. The skim milk and cream were then mixed to achieve a specified composition, in a process known as standardization. The standardized milk, which was stored at 4°C, was then directed to different dairy powder production processes to make products such as dry whole milk, non-fat dry milk, and dry dairy blends. 8 General introduction Figure 1.1 Schematic diagram of the evaporation and drying process (Scott et al., 2007). In the case of dry whole milk at this New Zealand whole powder factory described in Scott et al. (2007), the milk powder manufacturing runs were approximately 18 hours in length. To begin with, the temperature of the milk is increased by pushing it through a plate heat exchanger (PHE) and a direct steam injection (DSI). Following the heating treatment by PHE and DSI, the milk goes into evaporators. Following evaporation, the concentrated milk is sent to a scraped surface pre-heater and then undergoes homogenization before being dried and packed (Figure 1.1) (Scott et al., 2007). 1.2 Spoilage organisms associated with dairy powder products Dairy powder products (dairy-concentrate end products) are generally considered to be microbiologically stable, because of their low water content which prevents microbial cells from growing. However, bacterial spores may be present in the product. The ecology of spore formation is further discussed later in this chapter. After the powder is reconstituted with other products that have high water content, pre-existing spores can germinate and grow. This may result in enzyme and acid production, with the consequential development of an off-flavour, loss of structure, or coagulation in the end products (Chopra and Mathur, 1984; Chen et al., 2004). Thermophilic bacilli are associated with contamination in dairy-concentrate processing environments (Burgess et al., 2010), and they can out-compete other bacteria in dairy-concentrate processing lines where high temperatures are applied, 9 General introduction and are a primary concern in such facilities (Watterson et al., 2014). In the dairy industry, thermophilic sporeformers are usually enumerated at 55 °C on aerobic plate count agar. Those that have been isolated from dairy products at this temperature can be divided into two groups: obligatory thermophiles and facultative thermophiles (also known as thermo-tolerant microorganisms). According to literature the obligate thermophiles grow at temperatures in the range of 30°C to 72°C; typical examples include Anoxybacillus spp. and Geobacillus spp. (Flint et al., 2001; Ronimus et al., 2003; Scott et al., 2007). Facultative thermophiles can grow at both mesophilic and thermophilic temperatures (approximately 15°C - 65°C). Examples of facultative thermophiles include Bacillus licheniformis, Bacillus coagulans, Bacillus sporothermodurans, and Bacillus subtilis (Crielly et al., 1994; Flint et al., 2001; Ronimus et al., 2003, Scheldeman et al., 2005). Obligatory thermophilic bacilli are less of a concern since they generally do not grow at temperatures below 37°C, while dairy-based concentrates are usually stored at temperatures below 37°C. However, exceptions have been reported, for example, obligatory thermophilic bacilli Geobacillus stearothermophilus are considered to be responsible for the flat sour spoilage of evaporated milk, which is milk with lowered water content and has not been subject to the final drying process which transforms it into milk powder (Kalogridou-Vassiliadou, 1992; Olson and Sorrells, 1992). Facultative thermophilic bacilli are reported to be more involved in incidents of spoilage: strains of B. licheniformis are capable of producing a slimy extra-cellular substance that can affect the quality of pasteurized milk and cream; B. subtilis has been associated with ropiness in raw and pasteurized milk as well as the spoilage of UHT and canned milk products; B. coagulans have been connected to the spoilage of UHT and canned milk products due to their production of lactic acid (Burgess et al., 2010). Under favourable environmental conditions, evaporated milk may undergo flat sour spoilage when it contains viable spores capable of germinating and growing at both mesophilic and thermophilic temperatures, depending on the strain (Gordon et al., 1989). Table 1.1 presents the growth characteristics of several thermophilic bacilli. In this thesis, obligatory